When exogenous DNA, RNA or a nucleic acid molecule is introduced into a cell, the cell is said to be transfected, known previously as transformed. Various methods are known by which the transfecting nucleic acid molecule becomes a permanent part of the transformed cell's genome. Unless specialized methods are used, permanent transformation is usually the result of integration of the transforming nucleic acid into chromosomal DNA at a random location. The transfecting DNA, or nucleic acid molecule, can also be introduced into the cell on a plasmid that replicates autonomously within the cell and which segregates copies to daughter cells when the cell divides. Either way, the locus of the transfecting nucleic acid molecule, with respect to endogenous genes of the cell, is unspecified. Gene targeting is the general name for a process, whereby chromosomal integration of the transfecting DNA, at a desired genetic locus, is facilitated to the extent that permanently transfected cells having the DNA at that locus can be obtained at a useful frequency. Typically, the gene at the target locus is modified, replaced, or duplicated by the transforming or transfecting (donor) nucleic acid molecule. As such, it is desired to have a method for targeting specific genes in animals, such as non-human mammals, amphibians, and insects, especially Drosophila, since it is a well-known model organism.
Generally, the steps taken to achieve gene targeting are intended to increase the likelihood of chromosomal integration at the desired locus and to select for the desired integration events that have occurred (or select against undesired integration events). Without such steps, the desired integration might occur by chance, but with such a low frequency as to be undetectable.
Yeast (Saccharomyces cerevisiae) has been a useful organism for the development of gene targeting methods. Rothenstein, R. (1991) Methods in Enzymology 194:281-301, reviewed techniques of targeted integration in yeast. The normal yeast process of homologous recombination was shown to permit integration of transforming plasmid DNA having a segment of sequence homologous to a selected yeast gene. When a double-strand break was introduced within the homologous segment, transformation with the resulting linear DNA resulted in a 10-1000-fold increased incidence of integration at or near the break. The longer the region of homology on either side of the break, the greater the frequency of recombination at the desired locus. Strategies for gene replacement, gene disruption, and rescue of mutant alleles were described in the above article.
The studies of gene targeting in yeast have been facilitated by the fact that individual transformed cells can be isolated and grown in pure culture to any convenient amount. In addition, the short doubling time of yeast cells in culture has allowed researchers to observe events that occur with a low frequency and to study the genetics of those events within a convenient time scale. When working with complex multicellular organisms, the number of individuals which can be assessed for a genetic change, and the time scale required for observing patterns of inheritance, are both increased. To achieve practical gene targeting in such organisms, techniques were developed to increase the frequency of observable targeting events and to increase the efficiency of selection for desired events. Practical methods of gene targeting have been developed in the fruit fly, Drosophila melanogaster, and in the mouse, Mus musculus, however, such methods have not been applicable to a wider range of organisms. One of the methods for gene targeting in Drosophila relates to an ends-in procedure.
Transposons have been utilized for inducing gene targeting in Drosophila. A transposon is a class of nucleic acid sequence that can move from one chromosomal site to another. Gloor, G. B., et al. (1991) Science, 253:1110-1117, described utilizing the property of the P element transposon to generate a double strand gap when a transposition event occurs, the gap being located at the site formerly occupied by the transposon. Under most circumstances, the resulting gap is repaired by copying from homologous sequences on the sister chromatic. If a homologous sequence is present in the cell at an ectopic locus, for example on a plasmid, that sequence can also serve as a template to repair the double strand gap generated by the transposon's departure. This type of gap repair can then be employed to target a desired sequence to the locus of the departing transposon. The primary limitation of the process is that the host organism must have a transposon located at or near the target site.
The FLP-FRT recombinase system of yeast was employed to mobilize FRT-flanked donor DNA and generate re-integration at a different chromosomal location (Golic, M. M., et al., (1997) Nucl. Acids Res. 25:3665-3671). The donor DNA was introduced into the Drosophila chromosome flanked by repeats of the FRT recombinase recognition site, all within a P element for integration. The FLP recombinase was introduced under control of a heat-shock promoter, so that the enzyme could be activated by the investigators at a specified time. The action of FLP recombinase resulted in excision of the donor DNA followed by a second round of recombination at a target site where another FRT site was present. The phenomenon could be observed by using flies having the target FRT site at the locus of a known gene where an altered phenotype was detectable. This method is limited by the requirement of a target FRT site near a known gene.
Gene targeting in mammals has only been achieved, to any significant degree, in the mouse. Uniquely, in the case of the mouse, a pluripotent cell line exists, whereby embryonic stem (ES) cells can be grown in culture, transformed, selected, and introduced into an embryonic stage, preferably the blastocyst stage of the mouse embryo. Embryos bearing inserted transgenic ES cells develop as genetically chimeric (individuals composed of genetically different cells) offspring. By interbreeding siblings, homozygous mice carrying the selected genes can be obtained. An overview of the process and its limitations is provided by Capecchi, M. R. (1989) Trends in Genetics 5:70-76; and by Bronson, S. K. (1994) J. Biol. Chem. 269:27155-25158.
Both homologous and non-homologous recombination occurs in mammalian cells. Recombination is the occurrence of progeny with combinations of genes other than those that occurred in the parents, due to independent assortment or crossing over. Both homologous and non-homologous processes occur with low frequency and non-homologous recombination occurs more frequently than homologous recombination. ES cells are transfected with a DNA construct that combines a donor DNA having the modification to be introduced at the target gene site, with a flanking sequence homologous to the target site, and marker genes, as needed, for selection, as well as any other sequences that may be desired. The donor construct need not be integrated into the chromosome initially, but can recombine with the target site by homologous recombination, or at a non-target site by non-homologous recombination. Since these events are rare, dual selection is required to select for recombinants and to select against non-homologous recombinants. The selections are carried out in vitro on the ES cells in culture. PCR screening can also be employed to identify desired recombinants. The frequency of homologous recombination is increased as the length of the region of homology in the donor is increased, with at least 5 kb of homology being preferred. However, homologous recombination has been observed with as little as 25-50 bp of homology. Donor DNA, having small deletions or insertions of the target sequence, are introduced into the target with higher frequency than point mutations. Both insertions of sequence and replacement of the target, as well as duplication in whole or in part of the target, can be accomplished by appropriate design of the donor vector and the selection system, as desired, for the purpose of the targeting.
Gene targeting in mammals, other than the mouse, has been limited by a lack of ES cells capable of being transplanted and of contributing to germline cells of developing embryos. However, techniques related to cloning technology have opened new possibilities for gene targeting in other species. McCreath, K. J., et al. (2000) Nature 405:1066-1069, have reported successful targeting in sheep by carrying out transformation and targeting selection in primary embryo fibroblast cells. Fibroblast nuclei that were successfully targeted were then transferred to enunciated egg cells, followed by implantation in the uterus of a host mother. The technique provides the advantage that the generation of chimeric animals and subsequent breeding to homozygosity are not required. However, the time available for carrying out targeting and selection is comparatively short.
The use of recombinases and their recognition sites has proven to be a valuable tool once the initial targeting event has been achieved. For a review of the techniques applying the site-specific recombinase systems, see Sauer, B. et al. (1994), Current Opinion in Biotech. 5:521-527. See also U.S. Pat. No. 4,959,317. For example, repeated targeting at a given locus is facilitated by including recombination-specific recombination sites in the initial targeting construct. Once in place, the recombination sites can be used, in combination with the respective recombinases, to provide highly efficient transfer of an exogenous DNA to the locus of the recombination site. A recombinase system commonly used is the Cre recombinase, which recognizes a sequence designated loxP. The Cre recombinase and loxP recognition site are derived from bacteriophage P1.
Another widely used system, derived from the 2μ circle of Saccharomyces cerevisiae, is the FLP recombinase, which recognizes a specific sequence, FRT. In both systems, the effect of recombinase activity is determined by the orientation of the recognition sites flanking a given segment of DNA. A DNA sequence, flanked by directly repeated recombination sites and then integrated into the genome by either homologous or illegitimate recombination, can subsequently be removed simply by providing the corresponding recombinase. One useful consequence of this property has been exploited to remove an unwanted selection marker from the target site once homologous recombination has occurred and selection is no longer necessary. In another application, a gene which may exert a toxic effect can be maintained in a dormant state by inserting a lox-flanked sequence between the promoter and the gene, the sequence being designed to prevent expression of the gene. Expression of Cre activity results in excision of the intervening sequence and allows the promoter to act to activate the dormant gene. Cre can be introduced by mating or provided in an inducible form that permits activation at the investigator's control. A variety of other post-targeting strategies can be facilitated by the use of site-specific recombination systems, as known in the art.
As has been shown in yeast, introducing a double strand (ds) break into DNA increases recombination frequency. A number of studies have demonstrated that introducing a ds break into a target site increased recombination with a homologous donor DNA about 100-fold. The ds break was created by providing an I-SceI site in the target DNA, then introducing and expressing an I-SceI endonuclease, along with a donor DNA, homologous to the target. Using Chinese hamster ovary (CHO) cells, Sargent, R. G., et al. (1997) Mol. Cell. Biol. 17:267-277, described an experiment for testing crossovers between tandem repeats of an APRT gene, one of which carried an I-SceI site. The occurrence of homologous recombination could be measured by crossovers between the tandem APRT loci, which eliminated an intervening thymidine kinase (TK+) gene, or within different segments of the APRT gene, itself, based on the presence or absence in the progeny of certain mutations located in one of the tandem genes. A ds break was generated at the I-SceI site by introducing and expressing the I-SceI endonuclease carried on a separate expression vector and introduced by transformation. A similar type of demonstration was reported by Liang, F., et al. (1998) Proc. Natl. Acad. Sci. USA 95:5172-5177. Cohen-Tannoudji, M. et al. (1998) Mol. Cell. Biol. 18:1444-1448, described the use of an I-SceI site introduced into a target gene by conventional targeting. Once in place, other constructs could be introduced at the same target (“knocked in”) by a subsequent trans formation with a desired donor construct and transient expression of I-SceI endonuclease to introduce a ds-break at the target. The efficiency of the second targeting step was reportedly 100-fold greater than was observed for conventional targeting. The method had the disadvantage that an I-SceI site was required at the target site.
U.S. Pat. No. 5,962,327 describes the I-SceI endonuclease and its recognition site. The patent also discloses general strategies using I-SceI that can be attempted for the site-specific insertion of a DNA fragment from a plasmid into a chromosome. A diagram of site-directed homologous recombination in yeast is presented. It should be noted that this technique was shown only in yeast.
The difficulty of introducing a linear DNA molecule into germline cells hindered development of targeting techniques in Drosophila. Recently, a method to generate such a linear fragment in vivo was reported, accompanied by a demonstration of gene targeting (Rong and Golic, 2000). The particular form of gene targeting that was shown is termed ends-in or insertional targeting. This occurs when a DNA double-strand break (DSB) is made within a stretch of DNA that provides contiguous homology to the target locus, and results in the insertion of the extrachromosomal donor to generate a duplication of the targeted region. An alternative arrangement, where DSBs are provided at each end of a homologous segment, is termed ends-out targeting, and causes a segment of chromosome to be replaced with an introduced segment (FIG. 1). In mouse and in yeast, some studies show that ends-out targeting is less efficient than ends-in, while others indicate that the two types can be equally efficient (Hastings et al., 1993, Hasty et al., 1991, Thomas et al., 1987, and Deng et al., 1992). Although ends-out targeting has been successful and extremely useful in other model eucaryotes, doubts about its efficacy in Drosophila have been raised because of a previous failure to obtain targeting by this method (Bellaiche et al., 1999).
Ends-in and ends-out targeting refer to the two arrangements of donor DNA that can be used for gene targeting. Both methods have been used for targeted mutagenesis, but require different designs of the DNA used for targeting. Ends-in targeting has been successful in Drosophila, but an earlier attempt at ends-out targeting had failed. Ends-in and Ends-out targeting methods and constructs are readily distinguished. In particular, an ends-in construct has only one DSB site. In certain instances, the efficiency with which ends-in promotes targeting is inadequate. Additionally, ends-in is not well suited for use as a rescue construct.
In a previous attempt at ends-out targeting in Drosophila, Bellaiche et al. (1999) failed to recover gene targeting events when screening for ends-out disruption of the white gene. A consideration of that failure may provide useful insight into constraints on the use of gene targeting in Drosophila. Bellaiche and colleagues chose to drive FLP and I-SceI expression with the B2-tubulin promoter, a male germline-specific promoter that drives transcription in primary spermatocytes. When targeting events were recovered from males, the events may have occurred in mitotic, not meiotic cells, because the heat shock promoter that was used was limited in its activity to the earliest stages of spermatogenesis (Bonner et al., 1984; Golic and Golic, 1996). Meiotic cells of the male germline, in which meiotic recombination does not occur, may be even less favorable to targeting. Furthermore, although the B2-tubulin promoter drives transcription pre-meiotically, it was found that, with a B2-tubulin-promoted FLP gene that was constructed, translation of the mRNA was predominantly post-meiotic (Golic et al., 1997). If the same was true for the constructs made by Bellaiche et al., then the attempted targeting may have occurred primarily in post-meiotic spermatic.
A second difference is that a large non-homologous stretch of DNA was located on one end of the homologous segment. It is conceivable that this interfered with targeting. In mouse ES cell targeting, however, this does not pose a significant impediment to targeting efficiency, thus, the preferred explanation is that B2-tubulin-promoted expression of the recombinase and endonuclease for the failure to obtain targeting is favored.
As such, it is desired to have additional gene targeting methods for use in Drosophila. It is especially desired to have an ends-out method that will work in Drosophila. 